The long-term goal of this project is to elucidate the molecular mechanism(s) controlling stem cell proliferation and differentiation in response to developmental and physiological cues. Stem cells are a unique subset of precursor cells which are self-renewing and capable of giving rise to a wide variety of differentiated progeny. Furthermore, insufficient stem cell division can lead to birth defects, while uncontrolled division can lead to many types of cancer. Thus, stem cells have an enormous impact on the health, viability and the maintenance of all organisms. In Drosophila, subsets of neuronal stem cells, or neuroblasts, are quiescent upon larval hatching and resume cell division at specific developmental stages during larval growth; the timing of neuroblast proliferation is affected by mutations in two genes: trol and ana. ANA is required to repress premature neuroblast division and TROL is required to initiate neuroblast division inhibited by ANA. Indirect evidence suggests that ANA must be secreted by the glial cells in order to function. This hypothesis will be tested by constructing a gene for a secretion-defective ANA protein and determining if this gene can provide normal ana function. We will also examine ANA localization by immunoelectron microscopy, and bypass the specific cell cycle steps blocked by ANA by induced expression of various cell cycle genes. To understand how trol stimulates neuroblast activation, the trol gene will be isolated and the sequence of the TROL protein analyzed for similarities with known proteins in the database. These results will be supplemented with analysis of the spatial and temporal patterns of distribution of trol transcripts and TROL protein. Both trol and ana must eventually transmit their signals to the cell cycle machinery to control neuroblast proliferation. We will use biochemical and molecular approaches to identify proteins in the signaling pathways and genetic and molecular analysis to examine the role of the transcription regulator evenskipped in the trol pathway. Finally, potential coordinate control of neuroblast proliferation by ana and trol will be addressed by examining trol transcription and TROL localization in an ana mutant background. These studies will provide important insights into the mechanism by which proliferation of stem cells is controlled by developmental cues, and will generate new tools and reagents to probe further into these mechanisms.